The long-term objective of my laboratory is to understand how new genes with novel functions originate and how these molecular innovations contribute to the survival, adaptation, and evolution of organisms. Although it is well known that gone duplication plays an important role in the evolution of novel gone functions, the evolutionary forces and molecular mechanisms underlying functional changes of duplicated genes remain poorly understood. Here the ribonuclease (RNase) A gene superfamily of mammals is used as a model system to address the above questions, because (1) the superfamily includes many recently duplicated genes with distinct functions, (2) these functions can be assayed relatively easily in vitro, with the feasibility of analyzing mutant proteins generated by site-directed mutagenesis, (3) members of the superfamily are involved in immunity and cancer and are related to human health, and (4) substantial biochemical, structural, and functional information is available on the superfamily. Eight members of the superfamily are known in humans, and they are pancreatic RNase (or RNase 1), eosinophil-derived neurotoxin (EDN or RNase 2), eosinophil cationic protein (ECP or RNase 3), RNase 4, angiogenin (or RNase 5), RNase k6 (or RNase 6), RNase 7, and RNase 8. As enzymes, all of them can cleave phosphodiester bonds in RNA. But they have also evolved other functions and are involved in various physiological processes including digestion of dietary RNAs, angiogenesis, and host defenses. Our specific aims are (1) to test two competing theories on the origin of new gene function by functional characterization of the reconstructed common ancestral gene of the duplicated EDN and ECP genes of higher primates, which have antiviral and antibacterial activities, respectively; (2) to identify amino acid substitutions responsible for the antibacterial function of ECP; (3) to identify amino acid substitutions that led to the potent ribonuclease activity and antiviral activity in EDN; (3) to investigate the recent emergence of a bactericidal allelic form of the RNase 7 gene in humans and to test its potential role in human health and evolution; (5) to study the evolution of disulfide-bridging in RNase 8 among hominoids and the functional consequences caused activity by changes in disulfide bonds; and (6) to study the cause of the rapid evolution of primate angiogenic and to identify key amino acid residues necessary for the angiogenic activity in angiogenin.